Identification of twelve O-glycosylation sites in equine chorionic gonadotropin beta and equine luteinizing hormone ss by solid-phase Edman degradation.
Abstract: The O-glycosylation sites for equine LHss (eLHss) and eCGss were identified by solid-phase Edman degradation of four glycopeptides derived from the C-terminal region. Both subunits were O-glycosylated at the same 12 positions, rather than the 4-6 sites anticipated. These sites were partially glycosylated, with carbohydrate attachment ranging from 20% to 100% for eCGss and from 10% to 100% for eLHss. When the C-terminal peptide containing all but one of the O-linked oligosaccharides was removed by mild acid hydrolysis of either eLHss or eCGss, hybrid hormones could be obtained by reassociating eLHalpha,eFSHalpha, or eCGalpha with the truncated ss subunit derivatives. These hybrid hormones were identical in LH receptor-binding activity when des(121-149)eLHss or des(121-149)eCGss were combined with the same alpha subunit preparation. Thus, O-glycosylation appears to be responsible for the ss subunit contribution to the substantial difference in LH receptor-binding activity between eLH and eCG. Comparison of the equid LH/CGss sequences with those available for the primate CGss subunits indicated a greater conservation of glycosylation patterns in the former.
Publication Date: 2001-01-03 PubMed ID: 11133668DOI: 10.1095/biolreprod64.1.136Google Scholar: Lookup
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- Comparative Study
- Journal Article
- Research Support
- U.S. Gov't
- P.H.S.
Summary
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The research identifies the existence of twelve O-glycosylation sites in equine luteinizing hormone (eLHss) and equine chorionic gonadotropin (eCGss), rather than the previously believed 4-6 sites. These sites have varied amounts of carbohydrate attachment, influencing hormone functionality. The study also suggested that O-glycosylation is responsible for the difference in LH receptor-binding activity between eLH and eCG.
Identification of O-glycosylation sites
- O-glycosylation sites in eLHss and eCGss were identified using solid-phase Edman degradation of four glycopeptides extracted from the C-terminal region.
- Contrary to the expected 4-6 sites, twelve O-glycosylation sites were discovered in both eLHss and eCGss. This revealed that both hormones have the same number of glycosylation sites, indicating a similar structure.
Carbohydrate attachment percentages
- These 12 O-glycosylated positions had a variable amount of carbohydrate attachment, ranging from 10%-100% in eLHss and 20%-100% in eCGss.
- The variance in carbohydrate attachment percentages implies that the level of glycosylation could affect the hormonal activity and function of eLHss and eCGss.
Impact of O-glycosylation on hormone functionality
- The study found that by removing a peptide that carried all but one of the O-linked oligosaccharides through mild acid hydrolysis from either eLHss or eCGss, hybrid hormones could be created. These hybrid hormones retained identical LH receptor-binding activity.
- This suggests that O-glycosylation could be responsible for the difference in LH receptor-binding activity between eLH and eCG. Thus, the addition or subtraction of O-glycosylation sites could enable the creation of hormones with varied functionalities.
Comparison with primate CGss subunits
- The study also compared the equid LH/CGss sequences with those available for the primate CGss subunits.
- It concluded that the O-glycosylation patterns were better conserved in equid hormones than in primates, suggesting possible evolutionary conservation of structure and function in equid hormones.
Cite This Article
APA
Bousfield GR, Butnev VY, Butnev VY.
(2001).
Identification of twelve O-glycosylation sites in equine chorionic gonadotropin beta and equine luteinizing hormone ss by solid-phase Edman degradation.
Biol Reprod, 64(1), 136-147.
https://doi.org/10.1095/biolreprod64.1.136 Publication
Researcher Affiliations
- Department of Biological Sciences, Wichita State University, Wichita, Kansas 67260-0026, USA. bousfiel@twsuvm.uc.twsu.edu
MeSH Terms
- Amino Acid Sequence
- Animals
- Carbohydrate Conformation
- Carbohydrates / analysis
- Chorionic Gonadotropin / chemistry
- Chorionic Gonadotropin / metabolism
- Chorionic Gonadotropin / pharmacology
- Glycopeptides / chemistry
- Glycopeptides / metabolism
- Glycoprotein Hormones, alpha Subunit / chemistry
- Glycoprotein Hormones, alpha Subunit / metabolism
- Glycosylation
- Horses
- Hydrogen-Ion Concentration
- Hydrolysis
- Leydig Cells / drug effects
- Leydig Cells / metabolism
- Luteinizing Hormone / chemistry
- Luteinizing Hormone / metabolism
- Luteinizing Hormone / pharmacology
- Male
- Molecular Sequence Data
- Protein Multimerization
- Radioligand Assay
- Rats
- Receptors, LH / metabolism
- Testosterone / biosynthesis
Grant Funding
- AG15428 / NIA NIH HHS
- DK52383 / NIDDK NIH HHS
Citations
This article has been cited 6 times.- Lee SY, Byambaragchaa M, Choi SH, Kang HJ, Kang MH, Min KS. Roles of N-linked and O-linked glycosylation sites in the activity of equine chorionic gonadotropin in cells expressing rat luteinizing hormone/chorionic gonadotropin receptor and follicle-stimulating hormone receptor. BMC Biotechnol 2021 Sep 5;21(1):52.
- Ebeler M, Pilgram F, Wellhöfer T, Frankenfeld K, Franzreb M. First comprehensive view on a magnetic separation based protein purification processes: From process development to cleaning validation of a GMP-ready magnetic separator. Eng Life Sci 2019 Aug;19(8):591-601.
- Kara E, Dupuy L, Bouillon C, Casteret S, Maurel MC. Modulation of Gonadotropins Activity by Antibodies. Front Endocrinol (Lausanne) 2019;10:15.
- Cohen L, Bousfield GR, Ben-Menahem D. The recombinant equine LHβ subunit combines divergent intracellular traits of human LHβ and CGβ subunits. Theriogenology 2015 Jun;83(9):1469-76.
- Cahoreau C, Klett D, Combarnous Y. Structure-function relationships of glycoprotein hormones and their subunits' ancestors. Front Endocrinol (Lausanne) 2015;6:26.
- Byambaragchaa M, Kim SG, Park SH, Shin MG, Kim SK, Kang MH, Min KS. Production of Recombinant Single-Chain Eel Luteinizing Hormone and Follicle-Stimulating Hormone Analogs in Chinese Hamster Ovary Suspension Cell Culture. Curr Issues Mol Biol 2024 Jan 5;46(1):542-556.
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